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Cells 2015, 4(1), 56-70; doi:10.3390/cells4010056

ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting

1
Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA
2
Ph.D., Biocenter, University of Wuerzburg, Am Hubland, 97074 Würzburg, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Alexander E. Kalyuzhny
Received: 3 November 2014 / Accepted: 14 January 2015 / Published: 20 January 2015
(This article belongs to the Special Issue ELISPOT Research)
View Full-Text   |   Download PDF [1699 KB, uploaded 20 January 2015]   |  

Abstract

Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs. View Full-Text
Keywords: ELISPOT; Normal Distribution; software; spot size; gating; cytokines; IFN-γ; IL-2; IL-4; IL-5; IL-17; T cells; CD4; CD8 ELISPOT; Normal Distribution; software; spot size; gating; cytokines; IFN-γ; IL-2; IL-4; IL-5; IL-17; T cells; CD4; CD8
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Karulin, A.Y.; Karacsony, K.; Zhang, W.; Targoni, O.S.; Moldovan, I.; Dittrich, M.; Sundararaman, S.; Lehmann, P.V. ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting. Cells 2015, 4, 56-70.

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